Antimicrobial drugs are not only applied as medication, but are also widely used as growth promoting substances in veterinary medicine. In modern farming the legal use of veterinary pharmaceuticals as feed additive is common practice. These feed additives prevent illness of the animal, enhance its growth or enhance the feed efficiency. The compounds may be added to the feed or the drinking water of the animals. In some countries administration by injections of for example growth promoting substances is allowed, thus increasing the amount of antimicrobial drugs in human food. Of course illegal use of veterinary drugs may also occur.
The presence of antimicrobial drugs in food is a growing concern among consumers. Allergic reactions and effects on the intestinal human flora caused by antibiotics are well known. Another issue is the development of resistant bacteria. It is well known that misuse of antibiotics (i.e. administration whenever this is not required on medical grounds, or incomplete courses of treatment) is the most important cause of the development of antibiotic resistance. Also, the presence of antimicrobial compounds in food leads to the increase of drug resistant bacteria. Human pathogenic bacteria may thus develop resistance to antimicrobials, which are both used as feed additive and in human medicine. Another drawback of the use of antibiotics in feed as growth promoter is that antibiotics influence the production process of fermented meat products such as sausages in a negative way, due to the inhibition of starter cultures.
Most veterinary drugs enter the human food chain through meat from slaughter animals or through animal products such as milk and eggs.
Control on the presence of antimicrobial drugs in slaughter animals, animal products and food products, is an important issue in food industry. In most countries maximum residue limits or tolerance levels for drug residues in fresh meat and meat products have been established. For compounds that are not allowed there is a zero tolerance, such substances may never be present in animal products. For most drugs a withdrawal period is determined. This is the minimal period between the last treatment and the time of slaughter. During this period drug residues decrease to a level below the maximum residue limit. However, in some instances, even after the withdrawal period too high drug concentrations may still be present in the animal. This may be caused by natural individual differences in the metabolism, or because of disturbance of the excretion process of the drug due to illness. Finally, procedures may not have been applied correctly in the withdrawal periods. In most countries the legislation concerning veterinary drug residues in food products is maintained by using an official control program. In general, governmental institutes examine a certain percentage of the slaughtered animals for the presence of veterinary drug residues. In addition, slaughterhouses or supermarkets may also examine raw meat, meat products and organs such as kidney and liver.
Although many of the most important antimicrobial drugs are excreted via the urine, urine is so far hardly used as substrate for examining the presence or absence of antimicrobial drug residues in slaughter animals. Examples of antimicrobial animal drugs, which are excreted via the urine, are B-lactams such as amoxicillin, ampicillin, penicillin G and penicillin V, tetracyclines such as chlortetracycline and oxytetracycline, aminoglycosides such as gentamycin and streptomycin, and sulfonamides such as sulfamidine.
The amount of antimicrobial compounds in urine is indicative for the levels of antimicrobial compounds in the edible part of the animal, e.g. the muscle tissue and the liver. It is easy to understand that examining urine offers enormous advantages. For many species of animals it is quite straightforward to obtain urine samples since many animals have specific points in time during which urine is excreted.
The use of urine as a sample to test the presence of antimicrobial drugs in the meat, would allow the animal to be tested before slaughtering. This offers the farmer the possibility to check if the withdrawal period was finished as expected. If not, the farmer will decide to wait a few days before transporting the animal to the slaughterhouse. The economic losses in case an animal is determined positive for antibiotics after slaughtering are high. It is quite obvious that the whole control system for detecting antimicrobial drug residues in the human food chain will be more efficient if the animals are examined prior to rather than after slaughtering.
As an alternative also blood samples of animals can be used to determine the presence or absence of antimicrobial drug residues in slaughter animals. Both urine and blood samples can of course also be obtained after slaughtering of the animal. In that case the valuable meat parts do not have to be damaged in order to obtain a sample.
Antimicrobial residues can be detected using microbial inhibition tests (e.g. agar diffusion tests). Such methods are mainly used for examining milk and meat fluid and are described in GB A 1467439, EP 0005891, DE 3613794, CA 2056581, EP 0285792 and U.S. Pat. No. 5,494,805. These descriptions all deal with ready to use tests that make use of a test organism. The test organism is mostly imbedded in an agar medium, which may contain an indicator, a buffer solution, nutrients and substances to change the sensitivity for certain antimicrobial compounds in a positive or negative way.
Suitable test organisms are strains of Bacillus, Streptococcus and/or Escherdchia coli. In general the principle of the test is that when antibacterial compounds are present in a sample in a concentration sufficient to inhibit the growth of the test organism, the color of an acid/base or redox indicator be retained, while in the absence of inhibition the growth of the test organism is accompanied by the formation of metabolites that will change the color of the indicator. The currently available test methods are suitable for the detection of antimicrobial residues in many different types of samples. The problem however with these test methods is that detection of antimicrobial residues in urine and blood is not possible due to the presence of disturbing substances. These substances interfere with the test leading to false positive results.
Some methods of preparing urine and blood samples are known. The easiest method is diluting the sample with water (at least threefold). However it is obvious that diluting of the sample leads to a loss of sensitivity with the same factor.
Adjusting the pH of each urine or blood sample to a certain value partly eliminates the differences between the samples. In the case of urine the pH may differ considerably. These fluctuations are observed between species, between animals of the same species and from day to day for individual animals. For instance, due to differences in feed and the time required for obtaining the urine sample the pH may vary quite strongly between acidic and basic values. However, such methods cannot be used in practice, since it would be the most convenient to examine urine samples at the site of the farmer. Measuring the exact pH and titration of the sample to a certain pH value using acidic or alkaline solutions is much too complex in this situation. In this respect Erasmuson et al. (Analyst 123, 2497-2499 (1998)) have suggested the removal of bicarbonate from urine samples prior to analysis by acidic treatment to pH 5.5. The disadvantages of this approach are that many operations need to be performed such as addition of several aliquots of acid, measuring of the pH-value and allowing carbon dioxide to evolve and that the samples are diluted thereby decreasing the sensitivity. Further it was also observed that adjusting the pH does not give a satisfactory solution for the majority of the samples since false positive results still occur. Finally also by adjusting the pH, a major drawback again is the dilution of the sample.
It can be concluded that up to now no suitable test method for the detection of antimicrobial residues in urine and blood samples is available. The present methods are unreliable, time consuming and may lead to false positive results.